- Email: [email protected]
Inhibition by dibromothymoquinone of photosynthetic electron transfer in chloroplasts of differing ultrastructure
ARCHIVES
OF BIOCHEMISTRY
AND
Inhibition Electron
BIOPHYSICS
168, 594-600
by Dibromothymoquinone
Transfer
in Chloroplasts
D. G. BISHOP Plant
Physiology
Unit,
(19%)
CSZRO
Division University,
N. Ryde, Received
of Differing
AND
of Food
of Photosynthetic
W.
Ultrastructure
G. NOLAN
Research and School of Biological 2113, Sydney, Australia
December
Sciences,
Macquarie
3, 1974
The effect of the plastoquinone antagonist, dibromothymoquinone, on the photoreduction of ferricyanide and plastocyanin by maize mesophyll, maize bundle-sheath and Euglena gracilis chloroplasts has been investigated. Maximum inhibition of FeCN and plastocyanin reduction by mesophyll chloroplasts was obtained at dibromothymoquinone concentrations of 5 x lo-’ M. At higher concentrations dibromothymoquinone acted as an electron shuttle, increasing the rate of reduction of both substrates. In contrast, little inhibition of FeCN and plastocyanin reduction by bundle-sheath chloroplasts occurred at 5 x lo-’ M dibromothymoquinone, and above this concentration of inhibitor, the extent of inhibition increased, with no shuttle effect being observed. Euglena chloroplasts showed a response intermediate between that of mesophyll and bundle-sheath chloroplasts. The presence of a shuttle effect caused by dibromothymoquinone appears to be directly related to the presence of a proton pump in the chloroplast preparations. Plastocyanin is reduced by photosystem 2 alone and shows some of the properties of a class III electron acceptor, although the rates of reduction observed were much lower than those observed with lipophilic class III acceptors.
The introduction of dibromothymoquinone (DBMIB)’ as an inhibitor of photosynthetic electron transfer has provided much information on the mechanism of electron transfer in chloroplasts and the associated phosphorylation reactions. DBMIB is thought to act as a competitive inhibitor at the plastoquinone site between the two photosystems (1, 2). It markedly inhibits the photoreduction of acceptors designated as Class I (3) (such as NADP+ or methyl viologen) but has little effect on Class III acceptors (such as oxidized pphenylenediamine) (4). It can also act as an electron acceptor (5) and under certain conditions, transfer electrons across the chloroplast membrane to other acceptors such as ferricyanide (FeCN) (4). Most studies of the effect of DBMIB on
photosynthetic electron transfer have been carried out on isolated spinach chloroplasts, although its effect on photosynthetic electron transfer in pea chloroplasts (6), in cells of the red alga, Porphyridium cruentum, and the green alga, Chlorella pyrenoidosa, has been described (7). The present report is concerned with the effect of DBMIB on the reduction from water of FeCN and plastocyanin by chloroplasts with differing ultrastructure. Those studied were the granal mesophyll and agranal bundle-sheath chloroplasts of maize; and chloroplasts of the green alga, Euglena gracilis, which contain appressed lamellae not differentiated into the grana characteristic of most higher plant chloroplasts.
1 Abbreviations used: DBMIB, dibromothymoquinone, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, DCMU, 3-(3,Cdichlorophenyl)-l,l-dimethylurea; FeCN, ferricyanide.
Class II mesophyll chloroplasts, whole bundlesheath chloroplasts and bundle-sheath chloroplast fragments were isolated from greenhouse-grown maize plants (&a mays var. GH390) by the procedure of
MATERIALS
594 Copyright All rights
0 1975 by Academic of reproduction
Press, Inc.
in any form reserved.
AND
METHODS
EFFECT
OF DIBROMOTHYMOQUINONE
Anderson et al. (8). Chloroplasts were isolated from autotrophically-grown Euglena gracilis by passage of the cells through a French press (IO). The photoreduction of FeCN and plastocyanin was measured with an Aminco-Chance dual wavelength spectrophotometer as previously described (9, 11). Ail reactions were carried out in phosphate buffer of pH 7.5 and unless otherwise indicated, the concentration of plastocyanin in the reaction was 50 PM. Tungsten light, filtered with one Corning 2-60 red filter and one Corning l-69 infrared filter, was usea in all spectrophotometric measurements. The energy incident on the sample was 2.9 x IOK ergs cm-? s-l. DBMIB was dissolved in dimethylformamide and added to the reaction mixture such that the concentration of dimethylformamide remained at 1.33%. Control samples contained 1.33% dimethylformamide. RESULTS
The effect of increasing concentrations of DBMIB on the photoreduction of FeCN from water by maize mesophyll chloroplasts is shown in Fig. 1. The degree of inhibition reaches a maximum of about 85% at an inhibitor concentration of 5 x 1O-7 M; this is similar to the conditions for maximum inhibition in spinach and pea chloroplasts (2,4, 6). Above this concentration, the rate of FeCN reduction increases because DBMIB acts as an electron shuttle across the chloroplast membrane, being i
L
FIG. ‘1. Effect of DBMIB concentration on the photoreduction of FeCN and plastocyanin by maize mesophyll chloroplasts. The reaction mixture contained in 0.75 ml; chloroplasts (3 pg chlorophyll), 100 mM NaCl, 2.5 mM KHQO,, 5 mM MgCl,, 0.05% w/v bovine serum albumin, 50 mM phosphate buffer (pH 7.5), 10 mM methylamine, 333 PM FeCN, or 50 NM plastocyanin and DBMIB as indicated.
ON
CHLOROPLASTS
595
itself photochemically reduced and subsequently chemically reoxidized by FeCN (4, 5). The maximum shuttle rate occurs at a DBMIB concentration of 1 x 1O-5 M when activity of FeCN reduction is restored to about 50% of that of the control, but at higher concentrations the observed rate of FeCN reduction again decreases. The photoreduction of plastocyanin from water is also affected by DBMIB (Fig. 1) although little inhibition occurs at inhibitor concentrations below lo-’ M. At higher concentrations of DBMIB, its effect on plastocyanin reduction is similar to that on ferricyanide reduction. The maximum restoration of activity occurs at 1 x 10ms M DBMIB but in this case, the rate with plastocyanin is restored to that of the control sample. Evidence that DBMIB serves as an electron shuttle to plastocyanin is shown in Fig. 2. In the control sample, light causes an immediate reduction of plastocyanin at the maximum rate and the reaction ceases when the light is turned off. In the presence of DBMIB (1 x 10m5 M), there is a lag in both the commencement and attainment of the maximum rate of plastocyanin reduction following the onset of illumination, because DBMIB is first reduced and subsequently reoxidized by plastocyanin. When the illuminating light is turned off, plastocyanin reduction continues for a considerable period, until all the DBMIB has been reoxidized. The addition of DCMU inhibits the light dependent reactions completely. Figure 3 compares the effect of DBMIB on FeCN reduction by mesophyll chloroplasts and bundle-sheath chloroplast fragments of maize. The response of the two types of chloroplasts differs markedly. There is little inhibition of FeCN reduction by bundle-sheath chloroplast fragments at DBMIB concentrations below 1 x lo-’ M but above this level, inhibition increases steadily. No shuttle effect is, however, detectable at DBMIB concentrations of around 1 x 1O-5 M. The absence of the shuttle effect can be conveniently expressed by comparing the ratio of activity at inhibitor concentrations of 1 x 10e5 M and 5 x lo-’ M. In mesophyll chloroplasts
BISHOP AND NOLAN
DdM”
t
FIG. 2. Photoreduction of plastocyanin in the presence and absence of DBMIB. For details of spectrophotometer settings, see Ref. (13). The concentration of DCMU was 20 LLM. i
= c" P e
_
DBMIB
cont.
3. Effect of DBMIB concentration on the photoreduction of FeCN by maize mesophyll chloroplasts and bundle-sheath chloroplast fragments. FIG.
where a shuttle effect is observed this ratio for FeCN reduction is greater than unity (3.7 in data of Fig. 3) while in bundlesheath chloroplasts the ratio is less than unity (0.4 in data of Fig. 3). No shuttle effect of DBMIB on plastocyanin reduction by maize bundle-sheath chloroplast fragments is detectable, the ratio of activities at 1 x 1O-6 M and 5 x lo-’ M DBMIB, being less than unity (Table I). The different responses of maize mesophyll and bundle-sheath chloroplasts to DBMIB is not due to fragmentation of bundle-sheath chloroplasts during isolation, because no
DBMIB shuttle effect is observed in intact bundle-sheath chloroplasts (Table I). In addition the shuttle effect observed in mesophyll chloroplasts can be abolished by one passage of the chloroplasts through a French Press at 18000 psi (Table I). The effect of DBMIB on FeCN reduction by chloroplasts of the green alga Euglena gracilis is shown in Fig. 4. In this case the response to the inhibitor is intermediate between that observed with mesophyll and bundle-sheath chloroplasts. These is no inhibition of FeCN reduction by DBMIB concentrations below lo-’ M, but a marked shuttle effect is apparent at higher concentrations of the inhibitor, reaching a maximum at 1 x 10e6 M DBMIB. A shuttle effect similar to that observed with FeCN, can also be detected when plastocyanin is used as substrate (Table I). Figure 5 shows the effect of three treatments on the saturation curve for plastocyanin reduction by maize mesophyll chloroplasts. Passage of the cells through a French press results in fragmentation of the chloroplasts, with the loss of some photosystem 2 activity and release of endogenous plastocyanin (12). At low concentrations of exogenous plastocyanin, the rate of its reduction by chloroplasts treated in a French press is greater than that of the control chloroplasts, but saturation is reached at a substrate concentration of
EFFECT
OF DIBROMOTHYMOQUINONE
ON
TABLE ACTIVITY
OF DBMIB
AS AN ELECTRON
Chloroplast
I
SHUTTLE
Substrate
IN MAIZE
AND
bundle-sheath
fragments
Maize bundle-sheath Maize mesophyll Maize mesophyll French Euglena gracilis
i
= P
pressed
FeCN Plastocyanin FeCN FeCN FeCN F&N Plastocyanin
5
8 P 0 Eo3 i E
E 1
f ZL 0 DBYIB
cont.
FIG. 4. Effect of DBMIB concentration photoreduction of FeCN by Euglena gracilis plasts.
on the chloro-
about 15 PM in the treated sample, while the rate of plastocyanin reduction by the control sample continues to increase with increasing substrate concentration (Fig. 5). Treatment of chloroplasts with the polyene antibiotic, amphotericin B, results in the release of plastocyanin from its site in the membrane but with little apparent damage to the membrane structure (13). However this treatment has no effect on the saturation curve for plastocyanin reduction (Fig. 5). At low concentrations of plastocyanin, the addition of DBMIB (1 x 1O-5 M) results in a marked increase in the rate of plastocyanin reduction, presumably due to the shuttle effect of DBMIB, but as the substrate concentration is increased, the rate of reduction in the control sample approaches that of the DBMIB-treated chloroplasts. The effect of the uncoupler, methylam-
Euglena
CHLOROPLASTS
DBMIB
concentration
0
5 x 10~’
rmol
Maize
597
CHLOROPLASTS
4.1 3.2 “1 ii:1 2.5 1.8 1.8
(M)
1 X lo-’
substrate reduced min-’ mgchl-’ 3.4 2.9 1.7 2.5 2.5 1.2 0.7
2.0 1.6 1.5 3.6 2.1 3.0 2.3
Ratio of activity 1 X lo-‘M 5 X lo-‘M DBMIB 0.59 0.55 0.88 1.44 0.84 2.50 3.28
ine, on photoreductions by maize chloroplasts inhibited by DBMIB is shown in Table II. The rate of photoreduction of plastocyanin by maize mesophyll chloroplasts is not significantly increased by the uncoupler, and no effect is observed in the presence of 5 x 10m7 M DBMIB (concentration for maximum inhibition) or 1 x 10m5 M DBMIB (concentration for maximum shuttle effect). FeCN reduction by untreated mesophyll chloroplasts is stimulated 2.3fold by methylamine but this stimulatory effect is almost completely abolished in the presence of DBMIB. Reduction of FeCN by maize bundle-sheath chloroplast fragments is virtually unaffected by the addition of methylamine, either in the presence or absence of DBMIB. DISCUSSION
The results presented demonstrate that the effect of DBMIB on photosynthetic electron transfer varies between chloroplasts of differing ultrastructure. The internal chloroplast membranes of most higher plants are differentiated into grana (containing both photosystems 1 and 2) and stroma lamellae (containing only photosystem 1) (14). Maize mesophyll cholorplasts possess this type of structure and DBMIB inhibits the photoreduction of FeCN from water by interrupting the flow of electrons between the two photosystems, at the plastoquinone site (2). However, the inhibition of FeCN reduction is not complete, because FeCN can also accept electrons from photosystem 2 alone. Increasing
598
BISHOP 7
r
=
French
Press
_
AND
NOLAN
Amphotericin
B
_
DBMIB
6-
1
I
10
I 30
,
I 50
1 10
I
Plastocyanin
1 30 cont.
I
1 50
[I
k
18 30
10
polar
FIG. 5. Effect of treatment of maize mesophyll chloroplasts, by passage through press, or with amphotericin B or DBMIB, on the saturation curve for plastocyanin O--O, control samples: n 4, treated samples. TABLE EFFECT
OF METHYLAMINE
AND DBMIB
Chloroplasts
1 50
a French reduction.
II
ON PHOTOREDUCTION CHLOROPLASTS
Substrate
OF FERRICYANIDE
Methylamine (10rnM)
AND
PLASTOCYANIN
DBMIB 0
BY MAIZE
concentration 5 x 10-7
(M)
1 x 10-S
bmol substrate reduced min-’ mgchll’ Maize
mesophyll
Plastocyanin
Maize
mesophyll
FeCN
Maize
bundle-sheath
fragments
FeCN
the concentration of DBMIB above that necessary for maximum inhibition of FeCN reduction, produces a stimulation of FeCN reduction, resulting from the photoreduction of DBMIB which is in turn reoxidized by FeCN (4, 5). This reaction however continues to be due to photosystem 2 alone and activity is not restored to that of the control sample in which FeCN reduction is mediated by both photosystems. It is noteworthy also that the rate of FeCN reduction in the presence of 1 x 1Om6M DBMIB does not reach the high rates observed in the presence of typical Class III lipophilic acceptors (3), such as oxidized phenylene diamine (15). The photoreduction of plastocyanin from water is not markedly inhibited by concentrations of DBMIB below 1 x 10m7 M (Fig. l), because it is reduced by
+ + +
4.5 5.0 5.9 13.4 2.8 3.3
1.5 1.4 1.9 2.4 2.7 3.1
6.5 6.2 5.0 6.2 1.5 1.5
photosystem 2 alone and its reduction, in contrast to that of FeCN, is not affected by a break in electron transfer between the two photosystems (13). Some inhibition of plastocyanin reduction is observed at DBMIB concentrations around 5 x 1O-7 M, but as the inhibitor concentration is further increased, the rate of reduction of plastocyanin increases, due to a shuttle effect caused by DBMIB (Figs. 1 and 2). However, because its reduction only ever involves photosystem 2, the rate of plastocyanin reduction can be completely restored to that of the control (Fig. 1). The marked differences in the response of FeCN reduction to DBMIB, by granal mesophyll and agranal bundle-sheath chloroplasts (Fig. 3), is consistent with other variations in the properties of the two types
EFFECT
OF DIBROMOTHYMOQUINONE
of chloroplast. Maize bundle-sheath chloroplasts (either intact or fragmented) appear to reduce FeCN by photosystem 2 alone, as indicated by a low pH optimum for the reaction (16) which is characteristic of reductions involving photosystem 2 (5, 17), and its insensitivity to the inhibitor amphotericin B (13). Consequently, interruption of electron flow between the two photosystems by DBMIB does not affect FeCN reduction by maize bundle-sheath chloroplasts to the extent observed in mesophyll chloroplasts. In addition, DBMIB at concentrations around 1 x 10m5 M does not act as an electron shuttle in bundlesheath chloroplasts, and a continual increase in the degree of inhibition of FeCN reduction is observed. The absence of a shuttle effect could be due to the fact that the site of FeCN reduction by photosystem 2 in bundle-sheath chloroplasts is more exposed to the substrate than in mesophyll chloroplasts. It is also possible, however, that no shuttle effect is observed because a pH gradient is necessary for this effect (17-19) and such a proton pump is lacking in bundle-sheath chloroplast preparations (20). Treatment of mesophyll chloroplasts in a French press at high pressure, which destroys the granal structure, converts them to particles with properties similar to bundle-sheath chloroplasts with respect to the inhibition of FeCN reduction by DBMIB (Table I), a low pH optimum for FeCN reduction, and insensitivity of FeCN reduction to amphotericin B (13). Such treatment also destroys the proton pump in granal chloroplasts (20). The response of plastocyanin reduction by bundle-sheath chloroplasts to increasing DBMIB concentrations is similar to the response of FeCN reduction, and once again no shuttle effect is observed. Isolated chloroplasts of E. gracilis do not contain an intact electron transfer system, due to the loss of a soluble cytochrome f (21). Such chloroplasts reduce FeCN by photosystem 2 alone, as shown by a low pH optimum for the reaction and its insensitivity to amphotericin B (13). In these respects and in the relative insensitivity of FeCN reduction to DBMIB concentrations below 1 x lo-’ M, they resemble bundle-
ON
CHLOROPLASTS
599
sheath chloroplasts or mesophyll chloroplasts treated with the French press. However, at DBMIB concentrations around 1 x 10m5M, a shuttle effect on FeCN reduction can be demonstrated with Euglena chloroplasts. The presence of a proton pump has also been demonstrated in Euglena chloroplasts (22). Plastocyanin is reduced by photosystem 2 alone and although a hydrophilic protein, exhibits some properties similar to the lipophilic class III acceptors (3). Its reduction is unaffected by treatment of maize mesophyll chloroplasts with amphotericin B under conditions which give rise to complete inhibition of methyl viologen reduction (13). When added in substrate quantities, exogenous plastocyanin may not be primarily reduced at the site at which it occurs in the electron transfer chain, but rather accepts electrons before that site. This conclusion is based on the following facts. Firstly, the reduction of plastocyanin is not significantly sitmulated by the uncoupler, methylamine, under conditions where FeCN reduction is increased more than twofold, (Table II) indicating that its reduction does not involve the phosphorylation site I (15, 23) which would be expected if it were acting as a class I acceptor and accepting electrons at the site of endogenous plastocyanin in the electron transfer chain. Insensitivity to uncouplers is characteristic of Class III electron acceptors such as oxidized p-phenylenediamine (3, 23) and of FeCN when being reduced by reactions involving photosystem 2 only (5, 24). Secondly, in chloroplast preparations where FeCN is reduced by photosystem 2 alone (maize bundle-sheath chloroplasts, maize mesophyll chloroplasts passed through a French press, Euglena chloroplasts), the inhibition of plastocyanin reduction by increasing concentrations of DBMIB is identical to that of FeCN. Thirdly, passage of maize mesophyll chloroplasts through the French press, while abolishing the shuttle effect of DBMIB (Table I) and removing plastocyanin from its site in the membrane (12) greatly increases the rate of plastocyanin reduction at below saturating substrate levels (Fig. 5), and lowers the
600
BISHOP
AND
saturating concentration. This treatment evidently makes sites of reduction more readily available to the substrate although some photosystem 2 activity is destroyed and electron flow between the two photosystems is interrupted. Fourthly, modification around the plastocyanin site in the chain by treatment with amphotericin B, which removes enodgenous plastocyanin from the membrane (13) does not affect either the rate or substrate saturation curve for plastocyanin reduction (Fig. 5). It has also been shown that treatment of maize mesophyll chloroplasts with amphotericin B does not induce a rapid photooxidation of plastocyanin (13). Presumably this treatment does not modify the membrane structure sufficiently to allow ready access of reduced plastocyanin molecules to the site of oxidation, and by analogy insufficient oxidized plastocyanin could diffuse in and out of that site to sustain the rates of reduction observed in Fig. 5. Finally DBMIB, by acting as a shuttle, can greatly increase the rate of plastocyanin reduction at limiting substrate concentrations (Fig. 5), which should not be the case if plastocyanin were only to accept electrons at its normal site in the chain, near photosystem 1 and after the site of inhibition by DBMIB. ACKNOWLEDGMENTS The technical assistance of Vicki Home is gratefully acknowledged. Dibromothymoquinone was a gift from Prof. A. Trebst and plastocyanin from N. F. Tobin. REFERENCES 1. TREBST, A., HARTH, E., AND DRABER, W. (1970) iVaturforsch. 25b, 1157-1159. 2. B~HME, H., REIMER, S., AND TREBST, A. (1971) Naturforsch. 26b, 341-352.
Z. Z.
NOLAN 3. SAHA, S., OU~AKUL, R., IZAWA, S., AND GOOD, N. E. (1971) J. Biol. Chem. 246, 3204-3209. 4. IZAWA, S., GOULD, J. M., ORT, D. R., FELKER, P., AND GOOD, N. E. (1973) Biochim. Biophys. Acta 305, 119-128. 5. GOULD, J. M., AND IZAWA, S. (1973) Eur. J. Biochem. 37, 185-192. 6. NYUNT, U. T., AND WISKICH, J. T. (1973) Plant Cell Physiol. 14, 1099-1106. 7. BIGGINS, J. (1974) Fed. Eur. Biochem. Sot. Lett. 38, 311-314. 8. ANDERSON, J. M., BOARDMAN, N. K., AND SPENCER, D. (1971) Biochim. Biophys. Acta 245,253-258. Biochem. Biophys. 9. BISHOP, D. G. (1973) Arch. 154, 520-526. 10. BISHOP, D. G., BAIN, J. M., AND &MILLIE, R. M. (1973) J. Erp. Bat. 24, 361-375. 11. BISHOP, D. G. (1973) Biochem. Biophys. Res. Commun. 54, 816-822. 12. SANE, P. V., AND HAUSKA, G. A. (1972) Z. Naturforsch. 27b, 932-938. 13. NOLAN, W. G., AND BISHOP, D. G. (1975) Arch. Biochem. Biophys. 166, 323-329. 14. PARK, R. B., AND SANE, P. V. (1971) Annu. Rev. Plant Physiol. 22, 395-430. 15. OUITRAKUL, R., AND IZAWA, S. (1973) Biochim. Biophys. Acta 305, 105-118. 16. BISHOP, D. G., ANDERSER, K. S., AND SMILLIE, R. M. (1972) Plant Physiol. 50, 774-777. 17. GOULD, J. M., AND IZAWA, S. (1973) Biochim. Biophys. Acta 314, 211-223. 18. TREBST, A., AND REIMER, S. (1973) Biochim. Biophys. Acta 325, 546-557. 19. GOULD, J. M., AND IZAWA, S. (1974) Biochim. Biophys. Acta 333, 509-524. 20. ARNTZEN, C. J., DILLEY, R. A., AND NEUMANN, J. (1971) Biochim. Biophys. Acta 245, 409-424. 21. SMILLIE, R. M. (1968) in The Biology of Euglena (Buetow, D. E., ed.), Vol. 2, pp. l-54, Academic Press, New York. Biophys. Acta 245, 22. KAHN, J. S. (1971) Biochim. 144-150. 23. GOULD, J. M., AND ORT, D. R. (1973) Biochim. Biophys. Acta 325, 157-166. 24. TREBST, A., AND REIMER, S. (1973) Biochim. Biophys. Acta 305, 129-139.
OF BIOCHEMISTRY
AND
Inhibition Electron
BIOPHYSICS
168, 594-600
by Dibromothymoquinone
Transfer
in Chloroplasts
D. G. BISHOP Plant
Physiology
Unit,
(19%)
CSZRO
Division University,
N. Ryde, Received
of Differing
AND
of Food
of Photosynthetic
W.
Ultrastructure
G. NOLAN
Research and School of Biological 2113, Sydney, Australia
December
Sciences,
Macquarie
3, 1974
The effect of the plastoquinone antagonist, dibromothymoquinone, on the photoreduction of ferricyanide and plastocyanin by maize mesophyll, maize bundle-sheath and Euglena gracilis chloroplasts has been investigated. Maximum inhibition of FeCN and plastocyanin reduction by mesophyll chloroplasts was obtained at dibromothymoquinone concentrations of 5 x lo-’ M. At higher concentrations dibromothymoquinone acted as an electron shuttle, increasing the rate of reduction of both substrates. In contrast, little inhibition of FeCN and plastocyanin reduction by bundle-sheath chloroplasts occurred at 5 x lo-’ M dibromothymoquinone, and above this concentration of inhibitor, the extent of inhibition increased, with no shuttle effect being observed. Euglena chloroplasts showed a response intermediate between that of mesophyll and bundle-sheath chloroplasts. The presence of a shuttle effect caused by dibromothymoquinone appears to be directly related to the presence of a proton pump in the chloroplast preparations. Plastocyanin is reduced by photosystem 2 alone and shows some of the properties of a class III electron acceptor, although the rates of reduction observed were much lower than those observed with lipophilic class III acceptors.
The introduction of dibromothymoquinone (DBMIB)’ as an inhibitor of photosynthetic electron transfer has provided much information on the mechanism of electron transfer in chloroplasts and the associated phosphorylation reactions. DBMIB is thought to act as a competitive inhibitor at the plastoquinone site between the two photosystems (1, 2). It markedly inhibits the photoreduction of acceptors designated as Class I (3) (such as NADP+ or methyl viologen) but has little effect on Class III acceptors (such as oxidized pphenylenediamine) (4). It can also act as an electron acceptor (5) and under certain conditions, transfer electrons across the chloroplast membrane to other acceptors such as ferricyanide (FeCN) (4). Most studies of the effect of DBMIB on
photosynthetic electron transfer have been carried out on isolated spinach chloroplasts, although its effect on photosynthetic electron transfer in pea chloroplasts (6), in cells of the red alga, Porphyridium cruentum, and the green alga, Chlorella pyrenoidosa, has been described (7). The present report is concerned with the effect of DBMIB on the reduction from water of FeCN and plastocyanin by chloroplasts with differing ultrastructure. Those studied were the granal mesophyll and agranal bundle-sheath chloroplasts of maize; and chloroplasts of the green alga, Euglena gracilis, which contain appressed lamellae not differentiated into the grana characteristic of most higher plant chloroplasts.
1 Abbreviations used: DBMIB, dibromothymoquinone, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, DCMU, 3-(3,Cdichlorophenyl)-l,l-dimethylurea; FeCN, ferricyanide.
Class II mesophyll chloroplasts, whole bundlesheath chloroplasts and bundle-sheath chloroplast fragments were isolated from greenhouse-grown maize plants (&a mays var. GH390) by the procedure of
MATERIALS
594 Copyright All rights
0 1975 by Academic of reproduction
Press, Inc.
in any form reserved.
AND
METHODS
EFFECT
OF DIBROMOTHYMOQUINONE
Anderson et al. (8). Chloroplasts were isolated from autotrophically-grown Euglena gracilis by passage of the cells through a French press (IO). The photoreduction of FeCN and plastocyanin was measured with an Aminco-Chance dual wavelength spectrophotometer as previously described (9, 11). Ail reactions were carried out in phosphate buffer of pH 7.5 and unless otherwise indicated, the concentration of plastocyanin in the reaction was 50 PM. Tungsten light, filtered with one Corning 2-60 red filter and one Corning l-69 infrared filter, was usea in all spectrophotometric measurements. The energy incident on the sample was 2.9 x IOK ergs cm-? s-l. DBMIB was dissolved in dimethylformamide and added to the reaction mixture such that the concentration of dimethylformamide remained at 1.33%. Control samples contained 1.33% dimethylformamide. RESULTS
The effect of increasing concentrations of DBMIB on the photoreduction of FeCN from water by maize mesophyll chloroplasts is shown in Fig. 1. The degree of inhibition reaches a maximum of about 85% at an inhibitor concentration of 5 x 1O-7 M; this is similar to the conditions for maximum inhibition in spinach and pea chloroplasts (2,4, 6). Above this concentration, the rate of FeCN reduction increases because DBMIB acts as an electron shuttle across the chloroplast membrane, being i
L
FIG. ‘1. Effect of DBMIB concentration on the photoreduction of FeCN and plastocyanin by maize mesophyll chloroplasts. The reaction mixture contained in 0.75 ml; chloroplasts (3 pg chlorophyll), 100 mM NaCl, 2.5 mM KHQO,, 5 mM MgCl,, 0.05% w/v bovine serum albumin, 50 mM phosphate buffer (pH 7.5), 10 mM methylamine, 333 PM FeCN, or 50 NM plastocyanin and DBMIB as indicated.
ON
CHLOROPLASTS
595
itself photochemically reduced and subsequently chemically reoxidized by FeCN (4, 5). The maximum shuttle rate occurs at a DBMIB concentration of 1 x 1O-5 M when activity of FeCN reduction is restored to about 50% of that of the control, but at higher concentrations the observed rate of FeCN reduction again decreases. The photoreduction of plastocyanin from water is also affected by DBMIB (Fig. 1) although little inhibition occurs at inhibitor concentrations below lo-’ M. At higher concentrations of DBMIB, its effect on plastocyanin reduction is similar to that on ferricyanide reduction. The maximum restoration of activity occurs at 1 x 10ms M DBMIB but in this case, the rate with plastocyanin is restored to that of the control sample. Evidence that DBMIB serves as an electron shuttle to plastocyanin is shown in Fig. 2. In the control sample, light causes an immediate reduction of plastocyanin at the maximum rate and the reaction ceases when the light is turned off. In the presence of DBMIB (1 x 10m5 M), there is a lag in both the commencement and attainment of the maximum rate of plastocyanin reduction following the onset of illumination, because DBMIB is first reduced and subsequently reoxidized by plastocyanin. When the illuminating light is turned off, plastocyanin reduction continues for a considerable period, until all the DBMIB has been reoxidized. The addition of DCMU inhibits the light dependent reactions completely. Figure 3 compares the effect of DBMIB on FeCN reduction by mesophyll chloroplasts and bundle-sheath chloroplast fragments of maize. The response of the two types of chloroplasts differs markedly. There is little inhibition of FeCN reduction by bundle-sheath chloroplast fragments at DBMIB concentrations below 1 x lo-’ M but above this level, inhibition increases steadily. No shuttle effect is, however, detectable at DBMIB concentrations of around 1 x 1O-5 M. The absence of the shuttle effect can be conveniently expressed by comparing the ratio of activity at inhibitor concentrations of 1 x 10e5 M and 5 x lo-’ M. In mesophyll chloroplasts
BISHOP AND NOLAN
DdM”
t
FIG. 2. Photoreduction of plastocyanin in the presence and absence of DBMIB. For details of spectrophotometer settings, see Ref. (13). The concentration of DCMU was 20 LLM. i
= c" P e
_
DBMIB
cont.
3. Effect of DBMIB concentration on the photoreduction of FeCN by maize mesophyll chloroplasts and bundle-sheath chloroplast fragments. FIG.
where a shuttle effect is observed this ratio for FeCN reduction is greater than unity (3.7 in data of Fig. 3) while in bundlesheath chloroplasts the ratio is less than unity (0.4 in data of Fig. 3). No shuttle effect of DBMIB on plastocyanin reduction by maize bundle-sheath chloroplast fragments is detectable, the ratio of activities at 1 x 1O-6 M and 5 x lo-’ M DBMIB, being less than unity (Table I). The different responses of maize mesophyll and bundle-sheath chloroplasts to DBMIB is not due to fragmentation of bundle-sheath chloroplasts during isolation, because no
DBMIB shuttle effect is observed in intact bundle-sheath chloroplasts (Table I). In addition the shuttle effect observed in mesophyll chloroplasts can be abolished by one passage of the chloroplasts through a French Press at 18000 psi (Table I). The effect of DBMIB on FeCN reduction by chloroplasts of the green alga Euglena gracilis is shown in Fig. 4. In this case the response to the inhibitor is intermediate between that observed with mesophyll and bundle-sheath chloroplasts. These is no inhibition of FeCN reduction by DBMIB concentrations below lo-’ M, but a marked shuttle effect is apparent at higher concentrations of the inhibitor, reaching a maximum at 1 x 10e6 M DBMIB. A shuttle effect similar to that observed with FeCN, can also be detected when plastocyanin is used as substrate (Table I). Figure 5 shows the effect of three treatments on the saturation curve for plastocyanin reduction by maize mesophyll chloroplasts. Passage of the cells through a French press results in fragmentation of the chloroplasts, with the loss of some photosystem 2 activity and release of endogenous plastocyanin (12). At low concentrations of exogenous plastocyanin, the rate of its reduction by chloroplasts treated in a French press is greater than that of the control chloroplasts, but saturation is reached at a substrate concentration of
EFFECT
OF DIBROMOTHYMOQUINONE
ON
TABLE ACTIVITY
OF DBMIB
AS AN ELECTRON
Chloroplast
I
SHUTTLE
Substrate
IN MAIZE
AND
bundle-sheath
fragments
Maize bundle-sheath Maize mesophyll Maize mesophyll French Euglena gracilis
i
= P
pressed
FeCN Plastocyanin FeCN FeCN FeCN F&N Plastocyanin
5
8 P 0 Eo3 i E
E 1
f ZL 0 DBYIB
cont.
FIG. 4. Effect of DBMIB concentration photoreduction of FeCN by Euglena gracilis plasts.
on the chloro-
about 15 PM in the treated sample, while the rate of plastocyanin reduction by the control sample continues to increase with increasing substrate concentration (Fig. 5). Treatment of chloroplasts with the polyene antibiotic, amphotericin B, results in the release of plastocyanin from its site in the membrane but with little apparent damage to the membrane structure (13). However this treatment has no effect on the saturation curve for plastocyanin reduction (Fig. 5). At low concentrations of plastocyanin, the addition of DBMIB (1 x 1O-5 M) results in a marked increase in the rate of plastocyanin reduction, presumably due to the shuttle effect of DBMIB, but as the substrate concentration is increased, the rate of reduction in the control sample approaches that of the DBMIB-treated chloroplasts. The effect of the uncoupler, methylam-
Euglena
CHLOROPLASTS
DBMIB
concentration
0
5 x 10~’
rmol
Maize
597
CHLOROPLASTS
4.1 3.2 “1 ii:1 2.5 1.8 1.8
(M)
1 X lo-’
substrate reduced min-’ mgchl-’ 3.4 2.9 1.7 2.5 2.5 1.2 0.7
2.0 1.6 1.5 3.6 2.1 3.0 2.3
Ratio of activity 1 X lo-‘M 5 X lo-‘M DBMIB 0.59 0.55 0.88 1.44 0.84 2.50 3.28
ine, on photoreductions by maize chloroplasts inhibited by DBMIB is shown in Table II. The rate of photoreduction of plastocyanin by maize mesophyll chloroplasts is not significantly increased by the uncoupler, and no effect is observed in the presence of 5 x 10m7 M DBMIB (concentration for maximum inhibition) or 1 x 10m5 M DBMIB (concentration for maximum shuttle effect). FeCN reduction by untreated mesophyll chloroplasts is stimulated 2.3fold by methylamine but this stimulatory effect is almost completely abolished in the presence of DBMIB. Reduction of FeCN by maize bundle-sheath chloroplast fragments is virtually unaffected by the addition of methylamine, either in the presence or absence of DBMIB. DISCUSSION
The results presented demonstrate that the effect of DBMIB on photosynthetic electron transfer varies between chloroplasts of differing ultrastructure. The internal chloroplast membranes of most higher plants are differentiated into grana (containing both photosystems 1 and 2) and stroma lamellae (containing only photosystem 1) (14). Maize mesophyll cholorplasts possess this type of structure and DBMIB inhibits the photoreduction of FeCN from water by interrupting the flow of electrons between the two photosystems, at the plastoquinone site (2). However, the inhibition of FeCN reduction is not complete, because FeCN can also accept electrons from photosystem 2 alone. Increasing
598
BISHOP 7
r
=
French
Press
_
AND
NOLAN
Amphotericin
B
_
DBMIB
6-
1
I
10
I 30
,
I 50
1 10
I
Plastocyanin
1 30 cont.
I
1 50
[I
k
18 30
10
polar
FIG. 5. Effect of treatment of maize mesophyll chloroplasts, by passage through press, or with amphotericin B or DBMIB, on the saturation curve for plastocyanin O--O, control samples: n 4, treated samples. TABLE EFFECT
OF METHYLAMINE
AND DBMIB
Chloroplasts
1 50
a French reduction.
II
ON PHOTOREDUCTION CHLOROPLASTS
Substrate
OF FERRICYANIDE
Methylamine (10rnM)
AND
PLASTOCYANIN
DBMIB 0
BY MAIZE
concentration 5 x 10-7
(M)
1 x 10-S
bmol substrate reduced min-’ mgchll’ Maize
mesophyll
Plastocyanin
Maize
mesophyll
FeCN
Maize
bundle-sheath
fragments
FeCN
the concentration of DBMIB above that necessary for maximum inhibition of FeCN reduction, produces a stimulation of FeCN reduction, resulting from the photoreduction of DBMIB which is in turn reoxidized by FeCN (4, 5). This reaction however continues to be due to photosystem 2 alone and activity is not restored to that of the control sample in which FeCN reduction is mediated by both photosystems. It is noteworthy also that the rate of FeCN reduction in the presence of 1 x 1Om6M DBMIB does not reach the high rates observed in the presence of typical Class III lipophilic acceptors (3), such as oxidized phenylene diamine (15). The photoreduction of plastocyanin from water is not markedly inhibited by concentrations of DBMIB below 1 x 10m7 M (Fig. l), because it is reduced by
+ + +
4.5 5.0 5.9 13.4 2.8 3.3
1.5 1.4 1.9 2.4 2.7 3.1
6.5 6.2 5.0 6.2 1.5 1.5
photosystem 2 alone and its reduction, in contrast to that of FeCN, is not affected by a break in electron transfer between the two photosystems (13). Some inhibition of plastocyanin reduction is observed at DBMIB concentrations around 5 x 1O-7 M, but as the inhibitor concentration is further increased, the rate of reduction of plastocyanin increases, due to a shuttle effect caused by DBMIB (Figs. 1 and 2). However, because its reduction only ever involves photosystem 2, the rate of plastocyanin reduction can be completely restored to that of the control (Fig. 1). The marked differences in the response of FeCN reduction to DBMIB, by granal mesophyll and agranal bundle-sheath chloroplasts (Fig. 3), is consistent with other variations in the properties of the two types
EFFECT
OF DIBROMOTHYMOQUINONE
of chloroplast. Maize bundle-sheath chloroplasts (either intact or fragmented) appear to reduce FeCN by photosystem 2 alone, as indicated by a low pH optimum for the reaction (16) which is characteristic of reductions involving photosystem 2 (5, 17), and its insensitivity to the inhibitor amphotericin B (13). Consequently, interruption of electron flow between the two photosystems by DBMIB does not affect FeCN reduction by maize bundle-sheath chloroplasts to the extent observed in mesophyll chloroplasts. In addition, DBMIB at concentrations around 1 x 10m5 M does not act as an electron shuttle in bundlesheath chloroplasts, and a continual increase in the degree of inhibition of FeCN reduction is observed. The absence of a shuttle effect could be due to the fact that the site of FeCN reduction by photosystem 2 in bundle-sheath chloroplasts is more exposed to the substrate than in mesophyll chloroplasts. It is also possible, however, that no shuttle effect is observed because a pH gradient is necessary for this effect (17-19) and such a proton pump is lacking in bundle-sheath chloroplast preparations (20). Treatment of mesophyll chloroplasts in a French press at high pressure, which destroys the granal structure, converts them to particles with properties similar to bundle-sheath chloroplasts with respect to the inhibition of FeCN reduction by DBMIB (Table I), a low pH optimum for FeCN reduction, and insensitivity of FeCN reduction to amphotericin B (13). Such treatment also destroys the proton pump in granal chloroplasts (20). The response of plastocyanin reduction by bundle-sheath chloroplasts to increasing DBMIB concentrations is similar to the response of FeCN reduction, and once again no shuttle effect is observed. Isolated chloroplasts of E. gracilis do not contain an intact electron transfer system, due to the loss of a soluble cytochrome f (21). Such chloroplasts reduce FeCN by photosystem 2 alone, as shown by a low pH optimum for the reaction and its insensitivity to amphotericin B (13). In these respects and in the relative insensitivity of FeCN reduction to DBMIB concentrations below 1 x lo-’ M, they resemble bundle-
ON
CHLOROPLASTS
599
sheath chloroplasts or mesophyll chloroplasts treated with the French press. However, at DBMIB concentrations around 1 x 10m5M, a shuttle effect on FeCN reduction can be demonstrated with Euglena chloroplasts. The presence of a proton pump has also been demonstrated in Euglena chloroplasts (22). Plastocyanin is reduced by photosystem 2 alone and although a hydrophilic protein, exhibits some properties similar to the lipophilic class III acceptors (3). Its reduction is unaffected by treatment of maize mesophyll chloroplasts with amphotericin B under conditions which give rise to complete inhibition of methyl viologen reduction (13). When added in substrate quantities, exogenous plastocyanin may not be primarily reduced at the site at which it occurs in the electron transfer chain, but rather accepts electrons before that site. This conclusion is based on the following facts. Firstly, the reduction of plastocyanin is not significantly sitmulated by the uncoupler, methylamine, under conditions where FeCN reduction is increased more than twofold, (Table II) indicating that its reduction does not involve the phosphorylation site I (15, 23) which would be expected if it were acting as a class I acceptor and accepting electrons at the site of endogenous plastocyanin in the electron transfer chain. Insensitivity to uncouplers is characteristic of Class III electron acceptors such as oxidized p-phenylenediamine (3, 23) and of FeCN when being reduced by reactions involving photosystem 2 only (5, 24). Secondly, in chloroplast preparations where FeCN is reduced by photosystem 2 alone (maize bundle-sheath chloroplasts, maize mesophyll chloroplasts passed through a French press, Euglena chloroplasts), the inhibition of plastocyanin reduction by increasing concentrations of DBMIB is identical to that of FeCN. Thirdly, passage of maize mesophyll chloroplasts through the French press, while abolishing the shuttle effect of DBMIB (Table I) and removing plastocyanin from its site in the membrane (12) greatly increases the rate of plastocyanin reduction at below saturating substrate levels (Fig. 5), and lowers the
600
BISHOP
AND
saturating concentration. This treatment evidently makes sites of reduction more readily available to the substrate although some photosystem 2 activity is destroyed and electron flow between the two photosystems is interrupted. Fourthly, modification around the plastocyanin site in the chain by treatment with amphotericin B, which removes enodgenous plastocyanin from the membrane (13) does not affect either the rate or substrate saturation curve for plastocyanin reduction (Fig. 5). It has also been shown that treatment of maize mesophyll chloroplasts with amphotericin B does not induce a rapid photooxidation of plastocyanin (13). Presumably this treatment does not modify the membrane structure sufficiently to allow ready access of reduced plastocyanin molecules to the site of oxidation, and by analogy insufficient oxidized plastocyanin could diffuse in and out of that site to sustain the rates of reduction observed in Fig. 5. Finally DBMIB, by acting as a shuttle, can greatly increase the rate of plastocyanin reduction at limiting substrate concentrations (Fig. 5), which should not be the case if plastocyanin were only to accept electrons at its normal site in the chain, near photosystem 1 and after the site of inhibition by DBMIB. ACKNOWLEDGMENTS The technical assistance of Vicki Home is gratefully acknowledged. Dibromothymoquinone was a gift from Prof. A. Trebst and plastocyanin from N. F. Tobin. REFERENCES 1. TREBST, A., HARTH, E., AND DRABER, W. (1970) iVaturforsch. 25b, 1157-1159. 2. B~HME, H., REIMER, S., AND TREBST, A. (1971) Naturforsch. 26b, 341-352.
Z. Z.
NOLAN 3. SAHA, S., OU~AKUL, R., IZAWA, S., AND GOOD, N. E. (1971) J. Biol. Chem. 246, 3204-3209. 4. IZAWA, S., GOULD, J. M., ORT, D. R., FELKER, P., AND GOOD, N. E. (1973) Biochim. Biophys. Acta 305, 119-128. 5. GOULD, J. M., AND IZAWA, S. (1973) Eur. J. Biochem. 37, 185-192. 6. NYUNT, U. T., AND WISKICH, J. T. (1973) Plant Cell Physiol. 14, 1099-1106. 7. BIGGINS, J. (1974) Fed. Eur. Biochem. Sot. Lett. 38, 311-314. 8. ANDERSON, J. M., BOARDMAN, N. K., AND SPENCER, D. (1971) Biochim. Biophys. Acta 245,253-258. Biochem. Biophys. 9. BISHOP, D. G. (1973) Arch. 154, 520-526. 10. BISHOP, D. G., BAIN, J. M., AND &MILLIE, R. M. (1973) J. Erp. Bat. 24, 361-375. 11. BISHOP, D. G. (1973) Biochem. Biophys. Res. Commun. 54, 816-822. 12. SANE, P. V., AND HAUSKA, G. A. (1972) Z. Naturforsch. 27b, 932-938. 13. NOLAN, W. G., AND BISHOP, D. G. (1975) Arch. Biochem. Biophys. 166, 323-329. 14. PARK, R. B., AND SANE, P. V. (1971) Annu. Rev. Plant Physiol. 22, 395-430. 15. OUITRAKUL, R., AND IZAWA, S. (1973) Biochim. Biophys. Acta 305, 105-118. 16. BISHOP, D. G., ANDERSER, K. S., AND SMILLIE, R. M. (1972) Plant Physiol. 50, 774-777. 17. GOULD, J. M., AND IZAWA, S. (1973) Biochim. Biophys. Acta 314, 211-223. 18. TREBST, A., AND REIMER, S. (1973) Biochim. Biophys. Acta 325, 546-557. 19. GOULD, J. M., AND IZAWA, S. (1974) Biochim. Biophys. Acta 333, 509-524. 20. ARNTZEN, C. J., DILLEY, R. A., AND NEUMANN, J. (1971) Biochim. Biophys. Acta 245, 409-424. 21. SMILLIE, R. M. (1968) in The Biology of Euglena (Buetow, D. E., ed.), Vol. 2, pp. l-54, Academic Press, New York. Biophys. Acta 245, 22. KAHN, J. S. (1971) Biochim. 144-150. 23. GOULD, J. M., AND ORT, D. R. (1973) Biochim. Biophys. Acta 325, 157-166. 24. TREBST, A., AND REIMER, S. (1973) Biochim. Biophys. Acta 305, 129-139.